Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the ability of al eukaryotic cells to function, grw and communicate. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). We study the biogenesis of ER-derived transport vesicles, aiming to uncover the rules that govern efficient capture of cargo proteins and the biophysical basis of membrane transformation events that give rise to spherical protein carriers. We use the model organism, Saccharomyces cerevisiae, to study this problem using a combination of genetic, biochemical and in vivo imaging approaches. Vesicle formation encompasses three fundamental processes: deformation of the donor membrane into a spherical transport carrier; selective recruitment of cargo proteins into the nascent bud; and scission of the membrane to release the vesicle. Traditionally, these processes were thought to be driven by discrete functions of the COPII coat, but our recent findings suggest a more intimate connection between the different events that conspire to yield a vesicle. For example, lumenally-oriented cargo proteins likely oppose the action of the COPII coat in deforming the lipid bilayer; the specific cargo composition at individual ER exit sites can thus directly impact the efficiency of vesicle formation by altering membrane properties. Furthermore, the GTP cycle of the coat, which controls coat assembly, vesicle scission and coat shedding, is regulated in part by a partnership between the cargo adaptor protein, Sec24, and the COPII accessory protein, Sec16, implicating cargo proteins in modulation of the GTP cycle. Taken together, these new findings place the cargo molecules as central players in the process of vesicle formation in vivo, which makes sense if we consider that vesicle formation must be an adaptable process that permits cells to respond to the changes in specific cargo burdens associated with changing environmental and developmental conditions. The current research proposal consists of two specific aims. (1) To unravel the complexity of the GTP cycle of the COPII coat, probing the impact of alterations to the GTPase activity of the coat and elucidating whether cargo plays a direct role in this process. (2) To characterize the influence of cargo proteins on membrane biophysical properties that impact the ability of the COPII coat to deform the ER membrane into transport vesicles. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease that are impacted by defects in protein traffic within the secretory pathway.